MEMBRANE TOXICITY OF THE PROTEIN-KINASE-C INHIBITOR CALPHOSTIN-A BY AFREE-RADICAL MECHANISM

The effects of calphostin A on cytoplasmic calcium levels, receptor-me diated calcium release, and membrane input resistance were measured in neuroblastoma cells. Calphostin A is a lipophilic, light-sensitive pe rylenequinone that generates singlet oxygen when illuminated. It inhib its the activity of protein kinase C (IC50 = 250 nM), but only in the presence of light. Phorbol esters normally attenuate carbachol-evoked calcium release. This effect was blocked by simultaneous exposure to l ight and calphostin A (40 nM) for 30 min. At higher doses (0.5-1 muM) calphostin A also approximately doubled the resting calcium level and decreased cell input resistance by 51%. These toxic effects did not oc cur in the dark or after preincubation with the antioxidant alpha-toco pherol. These data support the hypothesis that the calphostins act by partitioning into the membrane and producing singlet oxygen and endope roxides which then irreversibly modify protein kinase C and other memb rane proteins and lipids.